Exposure to phenanthrene affects oocyte meiosis by inducing mitochondrial dysfunction and endoplasmic reticulum stress

Abstract Objectives Phenanthrene (PHE) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs), which is a widespread environmental contaminant. Various studies showed that PHE has adverse impacts on animals and human health. It has been shown that PHE exposure induced follicular atresia and endocrine dyscrasia in female mice. However, the potential mechanism regarding how PHE affects female reproductive system especially the oocyte quality has not been elucidated. Methods and Results In this study, we set up PHE exposure model and found that PHE exposure compromised oocytes maturation competence by inhibiting spindle assembly and chromosomes alignment. Moreover, PHE exposure induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress, leading to increased reactive oxygen species (ROS) and aberrant calcium levels in cytoplasm, eventually induced oxidative stress and DNA damage in oocytes. Furthermore, we found that oral administration of PHE caused the occurrence of oxidative stress and apoptosis in female ovary. In addition, the oocyte exhibited aberrant spindle morphology and failure of actin cap formation in metaphase II oocytes. Conclusions Taken together, our study demonstrated that mitochondrial dysfunction and ER stress‐induced oxidative stress and DNA damage are the major cause of poor oocyte quality after PHE exposure.

failure in women. 8 Meanwhile, the toxic effects of PAHs could transmit to subsequent generations. 9 Therefore, the adverse effect of PAHs on the human has drawn more considerable attention. 3,[10][11][12][13] Phenanthrene (PHE), a type of low-molecular-weight PAH, was widely distributed in environment. 14 Several studies have reported that the concentration of PHE was the highest of the 16 PAHs compounds in drinking water and air. 15,16 As with other PAHs, long-term exposure to PHE has negative effects on various systems in organisms. Orally and intraperitoneal injection of PHE in rats induced the occurrence of oxidative stress and inflammation in liver and lung. 15 Moreover, PHE could induce hepatic lipid metabolism disorder via changing the composition of gastrointestinal bacteria in mice. 17 In addition, long-term exposure to environmental levels of PHE reduced the number of sperm and Sertoli cells. PHE exposure increased the level of apoptosis in testicle cells and altered the expression levels of oestrogen receptor, androgen receptor and aromatic hydrocarbon receptor in male mice. 18 It has been shown that PHE exposure increased follicular atresia and inhibited ovarian development by disrupting the function of hypothalamus-pituitary-gonadal (HPG) axis and the steroidogenic pathway aquatic organisms. [19][20][21] In addition, gestational exposure to PHE caused reproductive disorders in F1 adult female mice, which exhibited by the increased number of antral follicles and reduced levels of luteinizing hormone (LH) and estradiol in serum. 22 Although the toxic effect of PHE on ovarian function and sex hormone secretion has been demonstrated, the effect and potential molecular mechanisms of PHE-induced deterioration of oocyte quality remain unclear.
Oocytes are arrested in the prophase of meiosis I and resume meiotic maturation in response to luteinizing hormone surge. After germinal vesicle breakdown (GVBD), spindle is organized at or near centre of oocyte, which subsequently migrates to oocyte cortex.
Shortly after, polar body extrusion occurs, which is accompanied by segregation of homologous chromosomes. Therefore, oocyte maturation is a perquisite for successful fertilization and embryonic development. 23 Aberrant microtubules assembly and chromosomes segregation cause aneuploidy and spontaneous abortion in mammals. 24,25 The normal distribution and function of organelles, such as mitochondria and endoplasmic reticulum (ER), are essential for oocyte maturation. Mitochondria not only regulates the energy metabolism, but also maintains the homeostasis of intracellular Ca 2+ . Increase of cytosolic Ca 2+ concentration disrupts oxidative phosphorylation and redox homeostasis, or opens the mitochondrial permeability transition pore, which further compromises mitochondrial function. 26 Mitochondrial dysfunction usually causes excessive accumulation of reactive oxygen species (ROS), and further induces the occurrence of oxidative stress, DNA damage and impairs developmental competence of oocytes. 23,27,28 ER is an important organelle that responsible for folding and assembly of secretory proteins. Accumulation of misfolded or unfolded proteins in ER can disrupt ER homeostasis and active ER stress. Activation of ER stress can trigger unfolded protein response (UPR), which further deteriorates oocyte quality. 29 In the present study, we determined the effect of PHE on oocyte quality by setting up in vitro and in vivo PHE exposure model. Our results showed that PHE exposure caused failure of oocyte maturation in vitro through inducing mitochondrial dysfunction, oxidative stress and ER stress, and further caused the occurrence of DNA damage. Moreover, by constructing PHE-exposed in vivo mice model, we found that PHE exposure induced occurrence of oxidative stress and apoptosis in ovary. Additionally, the quality of MII oocytes-derived from in vivo were reduced which exhibited by the aberrant cytoskeleton assembly. These data will expand our knowledge of how PHE deteriorates oocyte quality and ovarian function in mammals.

| Animals and feeding regimens
All mice were housed at controlled condition temperature (25 ± 2 C) and in 12:12-h light/dark cycle. Water and food were provided and free access throughout the study. All procedures were in accordance with the guidelines of the Institutional Animal Care and Use Committee at Zhejiang A&F University, China.
Thirty mice (body weight: 28-30 g/mice) were randomly divided into two groups. The mice were continuously oral gavage administration of PHE (dissolved in corn oil) at dose of 0 and 5 mg/kg/day 17 for 30 consecutive days. Metaphase II (MII) collection and culture: 5 IU human chorionic gonadotrophin (HCG) was injected at 48 hours after PMSG injection.

| Oocyte collection and culture
After 13-14 h, female mice were super-ovulated, and MII oocytes were collected from ampulla of the oviduct.

| Real-time quantitative PCR
Gene expression was determined by real-time quantitative PCR and calculated using the 2 ÀΔΔCT method. Total RNA was extracted from 50 oocytes using Dynabeads mRNA DIRECT Purification Kit (Invitrogen).
The first strand cDNA was prepared using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) and quantitative PCR (qPCR) was conducted using the Hieff qPCR SYBR Green Master Mix (Yeasen).

| Statistical analysis
At least three independent biological replicates were examined for analysis. The statistical analysis was performed with the GraphPad Prism software 8.0.1 (La Jolla, USA). All the analysis of data was F I G U R E 1 Phenanthrene (PHE) exposure impairs oocyte maturation. (A) Representative images of germinal vesicle breakdown and polar body extrusion oocytes in control and PHE-exposed oocytes (arrowheads: the oocytes underwent germinal vesicle breakdown (GVBD) and polar body extrusion in control group, but failed GVBD and polar body extrusion in PHE-treated group). Scale bar, 100 μm. (B) Percentage of GVBD was quantified in control and oocytes exposed to different concentrations of PHE (200, 300 and 400 μM). (C) Representative images of first polar body extruded-oocytes from control and PHA-exposed groups. Scale bar, 100 μm. Data are represented as mean ± SD from at least three independent experiments. ns p > 0.05, *p < 0.05 and **p < 0.01 performed by one-way analysis of variance with Turkey's test for multiple comparisons.

| PHE disturbs MI spindle assembly and causes chromosome misalignment during oocyte meiotic maturation
To evaluate the reason for defect in oocyte meiosis after exposure to PHE, we determined spindle assembly and chromosome alignment during oocyte maturation. As shown in Figure 2A-C, we observed that the area of spindles in treatment group was significantly higher than controls (1.0 ± 0.1, n = 35 versus 1.4 ± 0.3, n = 54, p < 0.001). Importantly, the control group oocyte presented a typical barrel-shaped spindle. Whereas the PHE-treated oocytes showed multipolar spindles, and quantitative results showed that the percentage of aberrant spindle morphology was significantly higher in PHE-treated group than control group (13.8% ± 3.7%, n = 150 versus 56.5% ± 17.4%, n = 100, p < 0.01).
During anaphase of oocyte meiosis, chromosomes were aligned neatly on the spindle equator by microtubules to avoid aneuploidy.
Our results showed that most of control oocytes exhibited a wellaligned chromosome on the equatorial plate. In contrast, the occurrence of chromosomes misalignment in PHE-treated group was obviously increased. To further quantitatively evaluate the misalignment of chromosomes, we measured the relative spread distance of chromosomes and found that the spread distance of chromosomes was significantly increased after PHE treatment (1.0 ± 0.2, n = 26 versus 1.5 ± 0.4, n = 36, p < 0.001; Figure 2D,E).
Interestingly, we found that TPX2 (Targeting Protein for Xklp2), which is an important protein in microtubule nucleation and stability, was significantly increased after PHE treatment (1.0 ± 0.4, n = 27 versus 2.2 ± 0.9, n = 46, p < 0.001; Figure 2F,G). Thus, our results suggested that the aberrant expression of TPX2 might be associated with aberrant microtubule nucleation and chromosomes misalignment after PHE exposure.

| PHE disturbs mitochondrial function in oocytes
To further examine the potential toxic mechanism of PHE in oocyte, the mitochondrial membrane potential was assessed by JC-1 staining, which was an important criterion for evaluating mitochondrial function and activity. We found that PHE treatment led to an increase in monomers and a reduction in aggregate in oocytes ( Figure 3A). The quantitative results, the fluorescent intensity of the aggregate/monomer, showed that the mitochondrial membrane potential in PHE treatment group was lower than controls (1.0 ± 0.3, n = 31 versus 0.4 ± 2.0, p < 0.001; Figure 3B

| PHE induces ROS generation and DNA damage in oocytes
ROS were generated and consumed by mitochondria, while mitochondrial dysfunction usually induces oxidative stress accompanied by the accumulation of ROS, which has negative effect on oocyte quality. Accordingly, 2 0 -7 0 dichlorofluorescin diacetate staining was used to measure the levels of ROS in oocytes. Expectedly, fluorescence intensity was significantly increased in PHE-exposed group compared to controls, indicating that PHE exposure led to accumulation of ROS in oocytes (1.0 ± 0.2, n = 22 versus 1.5 ± 0.3, n = 25, p < 0.001; Figure 5A,B).
Given that excessive ROS can induce DNA damage, we next examined whether PHE exposure induced DNA damage during oocyte maturation by staining γH2AX. As shown in Figure 5C,D, the γH2AX signal was significantly increased after PHE treatment  Data are represented as mean ± SD from at least three independent experiments. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001

| PHE causes oxidative stress and apoptosis in ovarian tissues and disturbs MII oocytes quality
To better understand the adverse effects of PHE on the female reproductive system, in vivo PHE exposure mice model was built by oral gavage administration for 30 days ( Figure 6A). We found that the ovarian weights of PHE treatment mice were significantly reduced (1.0 ± 0.1, n = 4 versus 0.8 ± 0.1, n = 4, p < 0.05; Figure 6B). To further explore the effect of PHE on the ovarian function, the mRNA expression levels of antioxidase gene Sod 1, apoptosis-related genes, Bax and Bcl 2 were detected. The result showed that mRNA level of Sod 1 and apoptotic gene Bax were markedly increased in PHEtreated group ( Figure 6C). These findings suggested that PHE exposure also induced oxidative stress and apoptosis in ovarian cells.
Subsequent to the first polar body extrusion and upon fertilization, the spindle of metaphase II was formed and played a critical role for fertilization. Therefore, we next investigated the effect of PHE exposure on cytoskeletal organization in MII oocytes. MII oocytes were obtained from the ampulla of the oviduct in PHE-exposed mice. We observed that the percentage of aberrant spindle was remarkably increased after PHE exposure (4.8% ± 6.7%, n = 50 versus 39.7% ± 6.7%, n = 50, p < 0.01; Figure 6D,E). Next, we determined the effect of PHE on cortical polarization after PHE treatment by staining with phalloidin-TRITC. As shown in Figure 6F,G, the fluorescence intensity of cortical actin was remarkably reduced after PHE-treatment (1.0 ± 0.3, n = 25, versus 0.7 ± 0.3, n = 35, p < 0.001). Meanwhile, the actin cap was disappeared in most of PHEtreated oocytes compared with that of control group (21% ± 3.4%, n = 40, versus 65.4% ± 3.4%, n = 45, p < 0.05; Figure 6H,I).

| DISCUSSION
PAHs generated from organic combustion in industrial produc- impacts on oocyte quality. In this work, we investigated the effects of PHE exposure on oocyte quality and ovarian function.
We first assessed the effect of PHE on oocyte maturation capacity in vitro. From the resumption of meiosis to maturation, the oocyte must undergo two landmark events: GVBD and PBE. 36 The result showed that PHE exposure led to the failure of GVBD and PBE, suggesting that PHE significantly affected oocyte maturation. The spindle was a crucial organelle in oocyte meiosis, and aberrant spindle frequently resulted in the failure of meiotic processes. It has been shown that TPX2 was necessary for spindle assembly; aberrant TPX2 expression usually disrupted spindle morphology. 37 Our results found that PHE exposure disrupted spindle assembly, accompanied by the increased expression of TPX2, indicating that PHE could induce abnormal microtubule assembly, and recruit more TPX2 to spindle for During oocyte maturation, cortical polarization was crucial event for spindle asymmetric positioning and asymmetric cell division. It has been well documented that small GTPase superfamily played crucial role in cell polarization establishment. 40,41 Mutations or knockdown of Cdc42, a key member of the GTPase superfamily, led to abnormal actin assembly and failure of cell polarity establishment, which in turn blocked spindle migration and asymmetric cell division. 42,43 Meanwhile, Actin nucleation factor Arp2/3 complex were also indispensable in microfilament nucleation and cortical polarization establishment. 44 Inhibition of Arp2/3 activity by CK666 blocked the formation of actin in MII oocyte and also caused rapid dissociation of spindle from cortex. 45,46 In the present study, we observed that the fluorescence intensity of actin in PHE exposure group was noticeably decreased than controls, and the actin cap was inconspicuous or disappeared. Meanwhile, we found that the frequency of abnormal MII spindle was noticeably increased after PHE exposure, indicating that PHE exposure could both inhibit the function of TPX2 and Arp2/3 complex in MII oocytes.
Many studies on PAHs exposure suggested that the toxicants impaired mitochondrial function. 38,47,48 To adapt intracellular energy metabolism and redox homeostasis, mitochondria required continual fusion and fission throughout oocyte maturation. 49,50 In Drp1-depleted oocytes, mitochondria were aggregated with other organelles, like ER, which disrupted the homeostasis of Ca 2+ and oocyte quality. 51 In addition, deletion of Mfn2 induced mitochondrial dysfunction, accompanied by the accumulation of ROS and increased apoptosis in Mfn2-lacking oocytes. 52 We found that, in this study, PHE treatment remarkably disrupted the balance of mitochondrial fusion and fission by inducing aberrant expression of Drp1 and Mfn2. Imbalance of mitochondrial fission and fusion usually accompanied by the occurrence of oxidative stress, disrupting in calcium homeostasis and mitochondrial membrane potential. [53][54][55][56][57] Consistent with this, we observed that PHE exposure remarkably increased ROS levels and intracytoplasmic Ca 2+ in PHE-exposed oocytes as well as decreased mitochondrial membrane potential in oocytes. Excessive accumulation of ROS in oocytes always led to DNA damage, 58,59 in line with this, we found that the level of DNA damage in PHE-exposed oocyte was significantly higher than controls.
ER was the major intracellular organelle for protein synthesis.
Dysfunction of ER induced the activation of UPR signalling pathways and ER stress, eventually blocked oocyte maturation. 29 Moreover, ER was the major intracellular Ca 2+ storage compartment, and disturbances in intracellular calcium homeostasis could induce dysfunction of ER. 60 Our result showed that PHE treatment had no effect on ER distribution, but the amount of ER was significantly increased. Meanwhile, the expression of Chop and ATF4 were significantly elevated. This may be caused by the massive enrichment of unfolded proteins in ER, which activated UPR signalling pathways and ER stress. Accordingly, our results indicated that PHE exposure could cause oxidative stress by inducing mitochondrial dysfunction and ER stress during oocyte maturation.
It has been abundantly demonstrated that PHE exposure led to increase of apoptosis in cells and tissues by increasing the activity of caspase 3 and caspase 9 enzymes and Bax expression. 14,18 And the apoptosis is most likely due to oxidative stress in tissues caused by PHE. 15,61 In line with this, our result demonstrated that PHE induced oxidative stress and apoptosis in ovary, which may explain the low quality of oocyte after exposure of PHE in vivo.

| CONCLUSION
Taken together, the current study indicated that PHE exposure caused ER stress and mitochondrial dysfunction by disrupting intracellular redox and Ca 2+ homeostasis, eventually inducing DNA damage F I G U R E 7 Mechanism of abnormal meiotic progression caused by PHE exposure. PHE exposure caused mitochondrial dysfunction by inducing the imbalance of mitochondrial dynamics and ER stress, which further resulted in the occurrence of DNA damage and reduced oocyte quality and apoptosis in oocytes (Figure 7). These findings expanded our knowledge of the influence of PHE on female reproductive capacity in mammals.

ACKNOWLEDGEMENT
We thank associate professor Kunlin Chen from the Institute of Animal Science, Jiangsu Academy of Agricultural Sciences for the assistance of antibodies.

CONFLICT OF INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article.